Nucleotide-binding oligomerization domain 2 (NOD2) is a NLR family member that functions as an intracellular sensor of small peptides (such as muramyl dipeptide, MDP) derived from the peptidoglycan (PGN) component of the bacterial cell wall. Activation of NOD2 occurring upon sensing of its ligand through its LRR domain is followed by NOD2 oligomerization and exposure of its CARD domain. This enables a physical interaction between NOD2 and a downstream adaptor molecule, receptor interacting serine-threonine kinase (RICK), that then results in lysine 63 (K63)-linked polyubiquitination of RICK. Ubiquitinated RICK then interacts with TGF-beta-activated kinase 1 (TAK1) and TRAF6 to cause activation of inhibitor of nuclear factor Ikk-gamma kinase followed by nuclear translocation of NF-kappaB subunits. The functional importance of NOD2 is underscored by the fact that polymorphisms in the LRR domain of the CARD15 gene encoding NOD2 are associated with Crohns disease and other immune disorders such as graft versus host disease (GVHD). However, despite extensive investigation, the molecular mechanisms by which such polymorphisms contribute to these diseases are not completely understood. A possible clue to the nature of these mechanisms comes from the fact that MDP activation of NOD2 can both positively and negatively regulate Toll-like receptor (TLR)-medaited inflammatory responses. For example, synergistic production of pro-inflammatory cytokine responses has been observed in human antigen-presenting cells (APCs) upon simultaneous stimulation of MDP and TLR ligands. This synergism could contribute to the control of the gastrointestinal commensal microflora that is necessary for the prevention of Crohns disease. It should be noted, however, that the above described capacity of NOD2 activation to augment innate immune responses is accompanied by the fact that such activation also has a negative effect on TLR signaling. Thus, we and others have shown that pre-activation of NOD2 by MDP induces tolerogenic pro-inflammatory cytokine responses in human APCs upon subsequent challenge with TLR ligands. In addition, other investigators have shown that such tolerogenic responses were not observed in human APCs from patients bearing Crohns-disease-associated NOD2 mutations. We have also found that systemic injection of MDP protects NOD2-intact mice from experimental colitis, but not NOD2- or RICK-deficient mice. Collectively, these data support the idea that MDP activation of NOD2 down-regulates innate immune responses to intestinal microflora and thus suggest that the absence of such regulation leads to increased susceptibility to Crohns disease. In our previous studies, we demonstrated that MDP activation of NOD2 has down-regulatory effects on multiple TLR signaling pathways. We now provide a mechanism for such down-regulation by showing that MDP activation of NOD2 induces IRF4-mediated inhibition of molecular events essential to the activation of NF-kappaB, namely K63-linked polyubiquitination of RICK and TRAF6. The chain of evidence supporting this conclusion consisted first of the fact that MDP pre-stimulation of huuman DCs led to binding of RICK to IRF4 and the binding of IRF4 to TRAF6 and MyD88. As shown in over-expression studies conducted in a cell line and more importantly in physiologic human DCs, these interactions set the stage for MDP-NOD2-induced IRF4 inhibition of K63-linked polyubiquitination of RICK and TRAF6; in addition, such inhibition could be linked to down-regulation of NF-kappaB activation by the demonstration that siRNA down-regulation of IRF4 reverses the negative effect on NOD2 signaling on K63-linked polyubiquitination and NF-kappaB activation. These in vitro studies were then supported by in vivo studies showing that protection from the development of TNBS-colitis by MDP administration is accompanied by greatly increased IRF4 expression and interaction with TRAF6 and MyD88 as well as inhibition of K63-linked polyubiquitination of RICK and TRAF6 in colonic cells and this is associated with greatly reduced NF-kappaB activation. A final step in the chain of evidence consisted of studies showing that administration of an IRF4-expressing plasmid to mice both prevented TNBS-colitis and reversed already established TNBS-colitis and that such administration was also associated with interactions between the administered IRF4 and RICK, TRAF6 and MyD88. These latter studies established that MDP activation of NOD2 in vivo was in fact acting via IRF4 to inhibit NF-kappaB activity. Overall, these data provide a mechanistic explanation of how MDP-NOD2 stimulation of APCs negatively regulates inflammatory responses induced by TLR ligands and therefore explain why defective NOD2 function could lead to excessive TLR responses in the gut that contribute to the pathogenesis of Crohns disease.